Humanized antibody amd process for preparing same

ABSTRACT

A humanized antibody is produced by process comprising the steps of: (a) selecting a specificity determining residue (SDR) of the complementarity determining region (CDR) of murine monoclonal antibody heavy chain and light chain variable regions; and (b) grafting said SDR to at least one of the corresponding amino acid sequences in human antibody variable regions.

FIELD OF THE INVENTION

The present invention relates to a process for preparing a humanizedantibody by grafting SDRs (specificity determining residues) in CDRs(complementary determining residues) of murine monoclonal antibody tohuman antibody and the humanized antibody prepared according to saidprocess.

BACKGROUND OF THE INVENTION

For preventing infectious diseases such as hepatitis B, there hasgenerally been used a method of administering immunoglobulins formed inblood plasma against a target antigen. However, the method has theproblems that the immunoglobulins generally have low specificity and maycontain contaminants.

Murine monoclonal antibody derived from mouse has been reported to havehigh affinity to antigen and is suitable for mass-production. However,repeated injection of murine monoclonal antibody induces an immuneresponse because the murine antibody is regarded as a foreign antigen inhumans (Shawler D. L. et al., J. Immunol., 135, 1530-1535(1985)).

Accordingly, numerous efforts have been made to generate “humanizedantibody” by: grafting the CDR (complementarity determining region) ofmurine monoclonal antibody variable region which directly binds toantigens, to a human antibody framwork (CDR-grafting method); andreplacing the amino acid residues of the human antibody framework region(FR) that influence the CDR conformation with the amino acid residues ofmurine monoclonal antibody. The humanized antibody thus obtainedmaintains the affinity and specificity of original murine monoclonalantibody, and minimizes HAMA(human anti-mouse antibody) response inhumans (Riechmann et al., Nature, 332, 323-327(1988); Queen C. et al.,Proc. Natl. Acad. Sci. USA, 86, 10029-10033(1989); Nakatani et al.,Protein Engineering, 7, 435-443(1994)). However, this humanized antibodystill causes problems when injected repeatedly into humans (Stephens etal., Immunology, 85, 668-674(1995); Sharkey et al., Cancer Research, 55,5935s-5945s(1995)).

Approximately 300 millions of world population carry hepatitis B virus(“HBV”) which may cause chronic infection, leading to cirrhosis andhepatocellular carcinoma (Tiollais P. and Buendia M. A., Sci. Am., 264,48(1991)). The HBV envelope consists of three proteins, major proteincontaining S antigen, middle protein containing S and pre-S2 antigens,and large protein containing S, pre-S2 and pre-S1 antigens (Neurath A.R. and Kent S. B., Adv. Vir. Res., 34, 65-142(1988)). These surfaceantigens have been known to play important roles in the process offorming antibodies against HBV in hepatitis patient. The pre-S1 region,in particular, is found on infectious viral particles (Heermann et al.,J. Virol., 52, 396-402(1984)) and plays a role in attachment to cellsurface infection (Neurath et al., Cell, 46, 429(1986); Pontisso et al.,Virol., 173, 533, (1989); Neurath et al., Vaccine, 7, 234(1989)). Thus amonoclonal antibody against the pre-S1 would be effective against viralinfection.

The present inventors have previously reported a murine monoclonalantibody (KR127) against HBV pre-S1 (Korean Patent No. 246128), a murinemonoclonal antibody KR127 gene encoding same (Korean Patent No. 250832)and a humanized antibody (HZKP127I) of KR127 prepared by CDR-graftingmethod (Korean Patent No. 246128).

The present inventors have further endeavored to develop a humanizedantibody having minimized adverse immune response (HAMA response) aswell as enhanced affinity to antigen, and found that HAMA response canbe reduced when the amino acid residues of CDR of mouse antibody arereplaced with those of human antibody.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide aprocess for preparing a humanized antibody which is expected to showlower HAMA response and has higher affinity than humanized antibody ofthe prior art.

It is another object of the present invention to provide a humanizedantibody prepared according to said process.

It is a further another object of the present invention to provide a DNAencoding the heavy chain or light chain of said antibody and a vectorcomprising said DNA.

It is a still further object of the present invention to provide amicroorganism transformed with said vector.

In accordance with one aspect of the present invention, there isprovided a process for preparing a humanized antibody comprising thesteps of: (a) selecting a specificity determining residue (SDR) of thecomplementarity determining region (CDR) of murine monoclonal antibodyheavy chain and light chain variable regions; and (b) grafting the aminoacid residues of said SDR to at least one of the corresponding aminoacid sequences in human antibody variable regions.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention taken inconjunction with the following accompanying drawings; which respectivelyshow:

FIG. 1: the procedure for constructing an expression vector of achimeric heavy chain;

FIG. 2: the nucleotide and amino acid sequence of the humanized heavychain variable region;

FIG. 3: the procedure for constructing an expression vector of achimeric light chain;

FIG. 4: the nucleotide and amino acid sequence of the humanized lightchain variable region;

FIG. 5: the affinity to antigen of a humanized antibody having a heavychain CDR mutant;

FIG. 6: the procedure for constructing an expression vector of thehumanized antibody; and

FIGS. 7 and 8: the results of analysis for MHC class II-binding peptidesequences in heavy chain variable regions of HuKR127 and light chainvariable regions of HuKR127, respectively, which are compared withHzKR127I, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The humanized antibody of the present invention may be prepared bygrafting the amino acid residues of SDR of murine monoclonal antibody tothe corresponding amino acid sequences in human antibody variableregions.

SDRs of the murine monoclonal antibody used in the present invention maybe determined by independently replacing each amino acid residue of CDRof the murine monoclonal antibody with alanine, selecting transformantswhich have lower affinity (k_(D)) to antigen than the original murineantibody and determining the replaced CDR amino acid residues of saidtransformants as SDRs.

Further, in order to enhance the affinity to antigen, the CDR residuesof a mouse antibody that increase the affinity and the frameworkresidues that influence the conformation of CDR loops may also begrafted to the corresponding sites of human antibody.

For example, the present invention describes a process for preparing ahumanized antibody for hepatitis B virus (HBV) pre-S1 by using murinemonoclonal antibody KR127 (Korean Patent No. 250832) as follows:

After selecting SDR amino acid residues, which play important roles inbinding with antigen, from CDR of the murine monoclonal antibody KR127heavy and light chains, chimeric heavy chain and chimeric light chaingenes may be prepared by combining either the variable region of KR127antibody heavy chain with the constant region (C_(γ) 1) of humanantibody or the variable region of KR127 antibody light chain with theconstant region (C_(κ)) of human antibody.

SDRs of the murine monoclonal antibody for HBV pre-S1 are determined byreplacing each amino acid residue of CDR HCDR1 (aa 31-35), HCDR2 (aa50-65) and HCDR3 (aa 95-102) of the heavy chain (SEQ ID NO: 2) and CDRLCDR1 (aa 24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain(SEQ ID NO: 4) of the murine monoclonal antibody KR127 with alanineaccording to the alanine scanning mutagenesis method and selecting theamino acid residues (SDRs) whose replacement with alanine bring aboutmore than 3 times reduction in the affinity to antigen (K_(D)) ascompared with the original murine antibody. Throughout this description,amino acid residue number is assigned according to Kabat numberingscheme (Kabat, E. A. et al, Sequences of Proteins of ImmunologicalInterest. National Institute of Health, Bethesda, Md., 1991).

Examples of preferred SDR include tryptophan at position 33 (it isrepresented as “Trp33”), Met34, and Asn35 of HCDR1; Arg50, Tyr52, andPro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murinemonoclonal antibody KR27 heavy chain; Leu27b, Tyr27d, Ser27e; Asn28,Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Val89,Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 of themurine monoclonal antibody KR127 light chain.

The humanized antibody of the present invention can be prepared bygrafting one or more SDRs determined as above onto the human antibodyheavy chain and/or light chain. The human antibody heavy chain which maybe used in the present invention is human heavy chain DP7-JH4 consistingof human immunoglobulin germline VH gene segment DP7 (Tomlinson et al.,J. Mol. Biol., 227, 776-798, 1992) and JH4 segment (Ravetch et al.,Cell, 27, 583-591, 1981). The human antibody light chain which may beused in the present invention is human light chain DPK12-JH4 consistingof human immunoglobulin germline VK gene segment DPK12 (Cox et al., Eur.J Immunol., 24, 827-836 (1994)) and JH4 segment (Hieter et al., J. Biol.Chem., 257, 1516-1522 (1982)).

The humanized heavy chain of the present invention may be prepared bygrafting at least one of Trp33, Met34, and Asn35 of HCDR1; Arg50, Tyr52,and Pro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murinemonoclonal antibody KR127 heavy chain to the corresponding amino acidsequences in human antibody heavy chain. The inventive humanized lightchain may be prepared by grafting at least one of Leu27b, Tyr27d,Ser27e; Asn28, Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 ofLCDR2; and Val89, Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 ofLCDR3 of the murine monoclonal antibody KR127 light chain to thecorresponding amino acid sequences in human antibody light chainDPH12-JK4.

Moreover, the affinity to antigen of the humanized antibody can beenhanced by the follow substitutions:

(a) the amino acid residue at position 32 in HCDR1 of the modified humanheavy chain DP7-JH4 by Ala;

(b) the amino acid residue at position 97 in HCDR3 of the modified humanheavy chain DP7-JH4 by Arg or Ala;

(c) the amino acid residue at position98 in HCDR3 of the modified humanheavy chain DP7-JH4 by Val; and

(d) the amino acid residue at position 102 in HCDR3 of the modifiedhuman heavy chain DP7-JH4 by Arg or Ala.

In addition, Ala71 and Lys73 in framework region 3 in the heavy chainvariable region of KR127, which affects the conformation of the CDRloop, may further be grafted to human heavy chain DP7-JH4. Also, Leu36and Arg46 in framework region 2 in the light chain variable region ofKR127, which affects conformation of CDR loop, may be further grafted tohuman light chain DPH12-JK4.

The heavy chain variable region of humanized antibody of the presentinvention has the amino acid sequence of SEQ ID NO: 2, preferablyencoded by the nucleotide sequence of SEQ ID NO: 1 and the inventivelight chain variable region of humanized antibody has the amino acidsequence of SEQ ID NO: 4, preferably encoded by the nucleotide sequenceof SEQ ID NO: 3.

The humanized antibody heavy chain and light chain of the presentinvention may be encoded by a gene comprising a nucleotide sequencededuced from the humanized antibody heavy chain and light chainaccording to the genetic code. It is known that several different codonsencoding a specific amino acid may exist due to the codon degeneracy,and, therefore, the present invention includes in its scope allnucleotide sequences deduced from the humanized antibody heavy chain andlight chain amino acid sequence. Preferably, the humanized antibodyheavy chain and light chain gene sequences include one or more preferredcodons of host cell.

The humanized antibody consisted of the humanized heavy chain HuKR127HCof the present invention and humanized light chain HZKR127I prepared byCDR-grafting has an affinity to antigen of about over 50 times higherthan that of the humanized antibody HZKR127I.

The humanized antibody consisting of the humanized heavy chain HuKR127KCof the present invention and humanized light chain HZKR127I prepared byCDR-grafting has an affinity to antigen equal to that of the humanizedantibody HZKR127I.

The genes of humanized antibody heavy chain and light chain thusprepared may be inserted to pdCMV-dhfrC-HAV6 vector (KCTC 10028BP) toobtain an expression vector pdCMV-dhfrC-HuKR127 which can express bothhumanized antibody heavy chain HuKR127HC and light chain HZKR127I. Theexpression vector of the present invention may be introduced intomicroorganism, e.g., E. coli DH5α according to a conventionaltransformation method to obtain transformants E. coliDH5α/pdCMV-dhfrC-HuKR127. The transformants E. coliDH5α/pdCMV-dhfrC-HuKR127 was deposited on Mar. 13, 2002 with the KoreanCollection for Type Cultures(KCTC)(Address: Korea Research Institute ofBioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon,305-333, Republic of Korea) under the accession number, KCTC 10198BP, inaccordance with the terms of Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

Meanwhile, CHO/HuKR127, CHO (Chinese hamster ovary) cell linetransfected with vector pdCMV-dhfrC-HuKR127, was deposited on Mar. 13,2002 with the Korean Collection for Type Cultures(KCTC) under theaccession number, KCTC 10199BP, in accordance with the terms of BudapestTreaty on the International Recognition of the Deposit of Microorganismfor the Purpose of Patent Procedure.

The humanized antibody HuKR127 of the present invention produced byculturing the CHO/HuKR127 cell line has a higher affinity to antigen andis expected to reduce HAMA (human anti-mouse antibody) response to agreater extent than the conventional antibody prepared according to theCDR-grafting method.

Accordingly, the humanized antibody of the present invention can be usedin preventing hepatitis B virus infection and treating chronic HepatitisB.

Thus, for preventing hepatitis B virus infection and treating chronicHepatitis B, a pharmaceutical formulation of the inventive humanizedantibody may be prepared in accordance with any of the conventionalprocedures.

The pharmaceutical composition of the present invention can beadministered via various routes including intravenous and intramuscularintroduction. It should be understood that the amount of the activeingredient actually administered ought to be determined in light ofvarious relevant factors including the condition to be treated, thechosen route of administration, the age, sex and body weight of theindividual patient, and the severity of the patient's symptom; and,therefore, the above dose should not be intended to limit the scope ofthe invention in any way.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

EXAMPLE 1 Preparation of Mouse/Human Chimeric Heavy Chain Gene

The gene encoding leader sequence and the γ1 constant region of thehuman antibody heavy chain were separately prepared by carrying out PCRusing pCMV-HKR127HC ((Korean Patent No. 246128, KCTC 0531BP) as atemplate and a primer set of Ryu94 (SEQ ID NO: 5) and HUR43-1 (SEQ IDNO: 6) or HUR46-1 (SEQ ID NO: 9) and HUR31 (SEQ ID NO: 10).

The gene encoding heavy chain variable region of the murine monoclonalantibody KR127 was prepared by carrying out PCR using pKR127H(KoreanPatent No. 250832, KCTC 0333BP) as a template and primers HUR44-1(SEQ IDNO: 7) and HUR45-1(SEQ ID NO: 8). Ryu94: 5′-GAG AAT TCA CAT TCA CGA TGTACT TG-3′ HUR43-1: 5′-CTG CTG CAG CTG GAC CTG ACT CTG GAC ACC ATT-3′HUR44-1: 5′-CAG GTC CAG CTG CAG CAG TCT GGA CCT GAA CTG-3′ HUR45-1:5′-TGG GCC CTT GGT GGA GGC TGC AGA GAC AGTGAC-3′ HUR46-1: 5′-GCC TCC ACCAAG GGC CCA TCG GTC TTC CCC CTG-3′ HUR31: 5′-CAG CGG CCG CTC ATT TAC CCGGGG ACA G-3′

Each PCR reaction was carried out using 10 ng of template, 1 μl of eachprimer (50 ppm), 0.5 μl of Pfu DNA polymerase (Promega), 4 μl of 2.5 mMdNTPmix and 5 μl of 10× Pfu reaction buffer solution. Afterpre-denaturation at 95° C. for 5 minutes, a PCR cycle was repeated 25times, the cycle being composed of: 95° C. for 30 sec., 50° C. for 30sec. and 72° C. for 45 sec. After annealing the DNA fragment obtained byusing primers Ryu94 and HUR43-1, the DNA fragment obtained by usingprimers HUR44-1 and HUR45-1, and the DNA fragment obtained by usingprimers HUR46-1 and HUR31 were recombined by conducting recombinant PCRusing primers Ryu94 and HUR31. The recombinant PCR reaction was carriedout using the same reaction buffer solution as used above. Afterpre-denaturation at 95° C. for 5 minutes, a PCR cycle was repeated 30times, the cycle being composed of: 95° C. for 30 sec., 50° C. for 30sec. and 72° C. for 60 sec., and finally, the extension reaction wascarried out at 72° C. for 5 min.

The chimeric heavy chain gene thus prepared was cleaved withEcoRI(GAATTC) and NdeI (GCGGCCGC) and inserted at the EcoRI/NdeI sectionof vector pcDdA (plasmid which is removed ApaI site in the multiplecloning site of pcDNA received from Invitrogen), to obtain vectorpcDdAchKR127HC (FIG. 1). The base sequence of the chimeric heavy chainvariable region gene (KR127VH) was confirmed by DNA sequence analysis(FIG. 2).

EXAMPLE 2 Preparation of Mouse/Human Chimeric Light Chain Gene

The gene encoding reader sequence and the constant region of the humanantibody light chain were each prepared by carrying out PCR usingpKC-dhfr-HKR127 (Korean Patent No. 2000-33008, KCTC 0529BP) as atemplate and a primer set of Ryu86 (SEQ ID NO: 11) and HUR48 (SEQ ID NO:12) or HUR51 (SEQ ID NO: 15) and CK1D (SEQ ID NO: 16).

The gene encoding light chain variable region of the murine monoclonalantibody KR127 was prepared by carrying out PCR using pKR127K (KoreanPatent No. 250832, KCTC 0334BP) as a template and primers HUR49 (SEQ IDNO: 13) and HUR50 (SEQ ID NO: 14). Ryu86: 5′-CAA AGC TTG GAA GCA AGA TGGATT CA-3′ HUR48: 5′-CAA GAT ATC CCC ACA GGT ACC AGA TAC-3′ HUR49: 5′-TGTGGG GAT ATC TTG ATG ACC CAA ACT-3′ HUR50: 5′-CAC AGA TCT TTT GAT TTC CAGCTT GGT-3′ HUR51: 5′-ATC AAA AGA TCT GTG GCT GCA CCA TCT-3′ CK1D: 5′-GCGCCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT TTG TGA CGG GCGAACTCAG-3′

Each PCR reaction was carried out according to the method described inExample 1 except that primers Ryu86 and CK1D were used to ligate theannealed DNA fragments obtained by PCR reactions.

The chimeric light chain gene thus prepared was cleaved with HindIII(AAGCTT) and XbaI (TCTAGA) and inserted at the HindIII/XbaI section ofvector pBluescript KS, to obtain a recombinant plasmid. Subsequently,the recombinant plasmind was cleaved with HindIII and ApaI and insertedat the HindIII/ApaI section of vector pCMV-dhfr (KCTC 8671P), to obtainplasmid pKC-dhfr-chKR127(FIG. 3). The base sequence of the chimericlight chain varible region gene (KR127VK) was confirmed by DNA sequenceanalysis (FIG. 4).

EXAMPLE 3 Mutation of CDR of Chimeric KR127 Antibody Heavy Chain byAlanine Injection

To examine whether each amino acid residue of KR127 heavy chain HCDR1(aa 31-35), HCDR2(aa 50-65) and HCDR3 (aa 95-102) binds to antigen, PCRreaction was carried out using vector pcDdA-chKR127HC as a template toprepare a modified gene, wherein an amino acid residue of CDR wasreplaced with alanine (the replaced amino acid residue No. was indicatedas Kabat number) (see FIG. 2).

A forward primer YM001N of SEQ ID NO: 17 was designed to provide thesequence corresponding to the reader sequence at the 5′-end of thechimeric heavy chain gene and EcoRI restrition site, and a reverseprimer YM003 of SEQ ID NO: 18 was designed to have the sequencecorresponding to the N-terminal downstream of CH1 domain of human heavychain gene and ApaI restriction site. YM001N: 5′-CCG GAA TTC ACA TTC ACGATG TAC TTG-3′ YM003: 5′-TGC CCC CAG AGG TGC T-3′

The 5′-end primer ym257 of SEQ ID NO: 19 (corresponding to nucleotideNos. 80 to 112 of SEQ ID NO: 1) was designed to replace Ser31 of HCDR1with alanine (S31A) and the 3′-end primer YM258 of SEQ ID NO: 20(corresponding to nucleotide Nos. 101 to 71 of SEQ ID NO: 1), to replaceAGT (coding for Ser) of nucleotide Nos. 91 to 93 of HCDRI gene with GCT(coding for alanine).

Each PCR reaction was carried out according to the method described inExample 1 except that primer sets, YM001N and Y4258; and ym258 andYM003, were used and also that primers YM001N and YM003 were used torecombine the annealed DNA fragments obtained by PCR.

The chimeric light chain gene thus prepared was cleaved with EcoRI andApaI and inserted at the EcoRI/ApaI section of vector pcDdA-chKR127HCprepared in Example 1, to obtain pcDdA-chKR127HC-S31A. The base sequenceof the humanized antibody heavy chain variable region gene was confirmedby DNA sequence analysis. Vectors containing mutants thus prepared areshown in Table 1.

In Table 1, primer and mutation positions are numbered based on the basesequence of SEQ ID NO: 1. TABLE 1 primer mutation CDR primer positionposition mutant vector HCDR1 F ym257  80-112 91-93 Ser(AGT)→pcDdA-chKR127HC-S31A R YM258 101-71  Ala(GCT) F ym259  83-112 94-96Ser(TCT)→ pcDdA-chKR127HC-S32A R YM260 106-73  Ala(GCT) F ym261  86-11797-99 Trp(TGG)→ pcDdA-chKR127HC-W33A R YM262 108-76  Ala(GCG) F ym263 90-118 100-102 Met(ATG)→ pcDdA-chKR127HC-M33A R YM264 111-79  Ala(GCG)F ym265  94-120 103-105 Asn(AAC)→ pcDdA-chKR127HC-N35A R ym266 112-81 Ala(GCC) HCDR2 F YM221 139-174 148-150 Arg(CGG)→ pcDdA-chKR127HC-R50A RYM222 158-128 Ala(GCC) F YM225 143-178 151-153 Ile(ATT)→pcDdA-chKR127HC-I51A R YM226 162-131 Ala(GCT) F YM227 145-180 154-156Tyr(TAT)→ pcDdA-chKR127HC-Y52A R YM228 165-135 Ala(GCT) F ym229 148-181157-159 Pro(CCT)→ pcDdA-chKR127HC-P52aA R YM230 167-136 Ala(GCT) F ym231150-186 160-162 Gly(GGA)→ pcDdA-chKR127HC-G53A R YM232 173-145 Ala(GCA)F ym233 152-188 163-165 Asp(GAT)→ pcDdA-chKR127HC-D54A R YM234 176-144Ala(GCT) F ym235 155-193 166-168 Gly(GGA)→ pcDdA-chKR127HC-G55A R YM236178-146 Ala(GCA) F ym237 158-194 169-171 Asp(GAT)→ pcDdA-chKR127HC-D56AR ym238 184-149 Ala(GCT) F ym239 160-195 172-174 Thr(ACT)→pcDdA-chKR127HC-T57A R ym240 185-150 Ala(GCT) F ym241 164-196 175-177Asn(AAC)→ pcDdA-chKR127HC-N58A R ym242 187-150 Ala(GCC) HCDR3 F YM207286-317 295-297 Glu(GAG)→ pcDdA-chKR127HC-E95A R YM208 305-274 Ala(GCG)F YM209 289-316 298-300 Tyr(TAC)→ pcDdA-chKR127HC-Y96A R YM210 307-276Ala(GCC) F YM211 292-318 301-303 Asp(GAC)→ pcDdA-chKR127HC-D97A R YM212313-279 Ala(GCC) F YM213 296-321 304-306 Glu(GAG)→ pcDdA-chKR127HC-E98AR YM214 315-285 Ala(GCG) F YM255 303-327 310-312 Tyr(TAC)→pcDdA-chKR127HC-Y102A R YM256 319-289 Ala(GGC)

TEST EXAMPLE 1 Expression of Chimeric Antibody Having a Modified HeavyChain and Its Affinity to Antigen

(Step 1) Expression of Chimeric Antibody

COS7 cells (ATCC CRL-165 1) were seeded to DMEM media (GIBCO) containing10% bovine serum and subcultured in an incubator at 37° C. under anatmosphere of 5% CO₂. 1×10⁶ cells thus obtained were seeded to the samemedia and incubated at 37° C. overnight. Thus, 5 μg of plasmidpKC-dhfr-chKR127 (expressing chimeric light chain) obtained in Example2, 5 μg of plasmid obtained in Example 3 were diluted withOPTI-MEMI(GIBCO) to 800 μl. 50 μl of Lipofectamine (GIBCO) were dilutedwith the same solution to 800 μl. The resulting solutions were added toa 15 μl tube, mixed and then, kept at room temperature for more than 15minutes. Meanwhile, COS7 cells incubated as above were washed threetimes with OPTI-MEM I. Then, 6.4 ml of OPTI-MEM I was added to theDNA-Lipofectamine mixture and the resulting solution was evenlydistributed on the COS7 cells, which were cultured for 48 hours in a 5%CO₂ incubator to obtain a supernatant. The resulting solution wassubjected to sandwich ELISA analysis using anti-human IgG (Sigma) as acapture antibody and anti-human antigen (Fc-specific)-horseradishperoxidase (PIERCE) as a secondary antibody to confirm the expression ofthe chimeric antibody.

(Step 2) Affinity to Antigen

150 ng of HBV recombinant antigen GST-pre-S1(1-56) (H. S. Kim and H. J.Hong, Biotechnology Letters, 17, 871-876(1995)) was coated to each wellof a microplate and 5 ng of the supernatant obtained in Step 1 was addedto each well. The resulting solution was subjected to indirect ELISAusing the same secondary antibody as used in step 1, followed bymeasuring the absorbance at 450 nm. Further, the affinity to antigen(K_(D)) of each modified heavy chain was determined by competitive ELISAmethod (Ryu et al., J. Med. Virol., 52, 226(1997)) and compared withthat of pCK-dhfr-chKR127 containing wildtype chimeric heavy chain. Theresult is shown in Table 2. TABLE 2 K_(D) CDR Mutant (nM) WT  11.0 ±1.664 H1 S31A 14.67 ± 2.386 S32A 8.455 ± 0.840 W33A >10000 M34A >10000N35A >10000 H2 R50A >10000 I51A 12.8 ± 1.05 Y52A 276.8 ± 23.60 P52aA170.3 ± 5.318 G53A 7.697 ± 0.980 D54A 1.663 ± 0.477 G55A 5.766 ± 0.211D56A 6.59 ± 1.09 T57A 13.68 ± 4.016 N58A 1.568 ± 0.085 H3 E95A >10000Y96A >10000 D97A 0.57 ± 0.03 E98A 64.2 ± 7.78 Y102A 3.581 ± 0.457

As shown in Table 2, the affinities to antigen of the mutants obtainedby replacing Trp33, Met34, or Asn35 of HCDR1; Arg50, Tyr52, or Pro52a ofHCDR2; Glu95, Tyr96, or Glu98 of HCDR3 with alanine were more than 3times lower than that of wild type. However; a mutant having alaninesubstituting for Asp97 or Tyr102 residue of HCDR3 exhibited an enhancedaffinity to antigen.

EXAMPLE 4 Preparation of HCDR3 Mutants and Their Affinities to Antigen

(Step 1) D97R and E98V Mutants

Each mutant was prepared by replacing Asp97 or Glu98 of HCDR3 witharginine as a positively charged amino acid (it is represented as“D97R”) or valine as a neutral amino acid (it is represented as “E98V”)according to the site-directed mutagenesis as used in Example 3. Vectorscontaining mutants prepared as above are shown in Table 3. TABLE 3primer mutation CDR primer position position mutant vector HCDR3 R P1312-279 301-303 Asp(GAC)→ pcDdA-chKR127HC-D97R F P2 295-326 Arg(CGG) RP3 312-279 301-303 Asp(GAC)→ pcDdA-chKR127HC-D97V F P4 295-326 Val(GTT)R P5 312-279 304-306 Glu(GAG)→ pcDdA-chKR127HC-E98R F P6 295-326 Arg(CGGR P7 312-279 304-306 Glu(GAG)→ pcDdA-chKR127HC-E98V F P8 295-326Val(GTT)

Then, each mutant thus obtained was measured for its affinity to antigenin according to the method described in Test Example 1 and compared withthat of the wild type.

As shown in FIG. 5, the affinity to antigen of D97R was more than 3times higher than that of the wild type, which the affinity to antigenof E98V, more than 4 times higher than that of the wild type. However,mutant E98R showed a low affinity to antigen.

(Step 2) D97R/E98V Mutant

To prepare D97R/E98V mutant containing both D97R and E98V, which werefound to be mutants having high affinity to antigen, PCR reaction wascarried out using pcDdA-chKR127HC-D97R which contains D97R gene as atemplate and primers P7 and P8.

Then, the D97R/E98V mutant thus obtained was measured for its affinityto antigen in according to the method described in Test Example 1.

As shown in FIG. 5, the affinity to antigen of D97R/E98V was more than15 times higher than that of the wild type.

(Step 3) D97R/E98V/Y102A Mutant

To prepare D97R/E98V/Y102A mutant containing D97R, E98V and Y102A, PCRreaction was carried out using pcDdA-chKR127HC-RV containing D97R/E98Vas a template and primers YM255 and YM256.

Then, the D97R/E98V/Y102A mutant (hereinafter “RVAA”) thus obtained wasmeasured for its affinity to antigen in according to the methoddescribed in Test Example 1.

As shown in FIG. 5, the affinity to antigen of D97R/E98V/Y102A wassimilar to that of D97R/E98V.

(Step 4) D97R/E98V/Y102E and D97R/E98V/Y102R Mutants

To prepare D97R/E98V/Y102E mutant and D97R/E98V/Y102R mutant, PCRreaction was carried out using pcDdA-chKR127HC-RV containing D97R/E98Vas a template, and primer sets P17/P18 and P19/P20, respectively.

Vctor containing mutants prepared above are shown in Table 4. TABLE 4primer mutation primer position position mutant vector HCDR3 R P17312-279 307-309 Tyr(TAC)→ pcDdA-chKR127HC-RVAE F P18 295-326 Glu(GAG) RP19 312-279 307-309 Tyr(TAC)→ pcDdA-chKR127HC-RVAR F P20 295-326Arg(CGT)

Then, D97R/E98V/Y102E mutant (hereinafter “RVAE”) and D97R/E98V/Y102Rmutant (hereinafter “RVAR”) thus obtained were measured for respectiveaffinities to antigen in according to the method described in TestExample 1.

As shown in FIG. 5, the affinity to antigen of RVAE was similar to thatof RVAA, while the affinity to antigen of RVAR was higher than that ofRVAA.

TEST EXAMPLE 2 Measurement of Affinity to Antigen of RVAR

The RVAR mutant prepared in step 4 of Example 4 was subjected tocompetitive ELISA to measure its affinity to antigen as follows:

COS7 cells were transfected with the plasmid prepared in step 4 ofExample 4 and the plasmid expressing chimeric lightchain(pKC-dhfr-chKR127) prepared in Example 2 to produce an antibody. 5ng of the antibody thus obtained was reacted with pre-S1 antigen (1×10⁻⁷to 1×10⁻¹² M) at 37° C. for 2 hours. The resulting solution was added toeach well of a 96-well microplate coated with pre-S1 antigen and reactedat 37° C. for 30 minutes, and then the resulting solution was subjectedto ELISA analysis according to the method described in Example 4. Usedas a control is chimeric antibody (chKR127) obtained from COS7 cellstransfected with pcDdA-chKR127HC and pKC-dhfr-chKR127.

The affinity to antigen of RVAR was about 1.8×10⁻⁰ M, which is 45 timeshigher than that of chKR127, about 8.2×10⁻⁹ M

EXAMPLE 5 Mutation of CDR of Chimeric KR127 Antibody Light Chain byAlanine Injection

To examine the affinity of each amino acid residue of KR127 light chainLCDR1 (aa 24-34), LCDR2(aa 50-60) and LCDR3 (aa 89-97) to antigen, PCRreaction was carried out using vector pKC-dhfr-chKR127 as a template toprepare a modified gene having each amino acid residue of CDR replacedwith alanine (the replaced amino acid residue Number was indicated asKabat number)(see FIG. 2).

Forward primer YM004 of SEQ ID NO: 21 was designed to provide thesequence corresponding to the reader sequence at the 5′-end of thechimeric light chain gene and the HindIII restrition site, and a reverseprimer YM009 of SEQ ID NO: 22 was designed to have the sequencecorresponding to the N-terminal region of human light chain gene and theBsiWI(CGTACG) restriction site. These primers were used in preparationof mutants of light chain CDR residue. YM004: 5′-CCA AAG CTT GGA AAG ATGGAT TCA CAG-3′ YM009: 5′-GCA GCC ACC GTA CGT TTG ATT TCC ACC TTG GT-3′

Forward primer YM135 was designed to replace Ser26 of LCDR1 with alanine(S26A) and a reverse primer YM136, to replace AGT coding for Ser at thenucleotide Nos. 76 to 78 of LCDRI gene with GCT coding for alanine.

PCR reactions were carried out according to the method described inExample 1 except that primer sets, YM004/YM135, and YM136/YM009, wereused and that primers YM004 and YM009 were used to recombine theannealed DNA fragments obtained by PCR.

The variable region gene of the mutant thus prepared was cleaved withHindIII and BsiWI and inserted at the HindIII/BsiWI section of vectorpKC-dhfr-chKR127, to obtain pKC-dhfr-chKR127BS-S26A. The base sequenceof the modified chimeric light chain variable region gene was confirmedby DNA sequence analysis. The vectors containing mutants prepared aboveare shown in Table 5.

In Table 5, the primer and mutation positions are numbered based on thebase sequence of SEQ ID NO: 3. TABLE 5 primer mutation primer positionposition mutant vector LCDR1 F YM135  67-102 76-78 Ser(AGT)-pKC-dhfr-chKR127BS-S26A R YM136 86-54 Ala(GCT) F YM137  69-107 79-81Gln(CAG)- pKC-dhfr-chKR127BS-Q27A R YM138 91-56 Ala(GCG) F YM139  70-11182-84 Ser(AGC)- pKC-dhfr-chKR127BS-S27aA R YM140 94-58 Ala(GCC) F YM141 73-114 85-87 Leu(CTC)- pKC-dhfr-chKR127BS-L27bA R YM142 98-64 Ala(GCC)F YM143  73-116 88-91 Leu(TTA)- pKC-dhfr-chKR127BS-L27cA R YM144 102-68 Ala(GCA) F YM145  79-118 91-93 Tyr(TAT)- pKC-dhfr-chKR127BS-Y27dA RYM146 103-69  Ala(GCT) F YM147  83-119 94-96 Ser(AGT)-pKC-dhfr-chKR127BS-S27eA R YM148 107-69  Ala(GCT) F YM149  84-120 97-99Asn(AAT)- pKC-dhfr-chKR127BS-N28A R YM150 110-70  Ala(GCT) F YM151 88-127 100-102 Gly(GGA)- pKC-dhfr-chKR127BS-G29A R YM152 114-74 Ala(GCA) F YM153  91-130 103-105 Lys(AAA)- pKC-dhfr-chKR127BS-K30A RYM154 116-77  Ala(GCA) F YM155  93-132 106-108 Thr(ACC)-pKC-dhfr-chKR127BS-T31A R YM156 118-80  Ala(GCC) F YM103  99-133 109-111Tyr(TAT)- pKC-dhfr-chKR127BS-Y32A R YM104 120-83  Ala(GCT) F N34A-F106-132 115-118 Asn(AAT)- pKC-dhfr-chKR127BS-Y34A R N34A-R 126-100Ala(GCT) LCDR2 F YM129 151-188 163-165 Leu(CTG)- pKC-dhfr-chKR127BS-L50AR YM130 175-140 Ala(GCG) F YM131 153-191 166-168 Val(GTG)-pKC-dhfr-chKR127BS-V51A R YM132 179-145 Ala(GCG) F YM133 157-192 169-171Ser(TCT)- pKC-dhfr-chKR127BS-S52A R YM134 181-147 Ala(GCT) F K53A-F163-187 172-174 Lys(AAA)- pKC-dhfr-chKR127BS-K53A R K53A-R 178-154Ala(GCA) F L54A-F 163-189 175-177 Leu(CTG)- pKC-dhfr-chKR127BS-L54A RL54A-R 180-159 Ala(GCG) F D55A-F 170-195 178-180 Asp(GAC)-pKC-dhfr-chKR127BS-D55A R D55A-R 184-163 Ala(GCC) F K56A-F 175-198181-183 Ser(TCT)- pKC-dhfr-chKR127BS-S56A R K56A-R 190-168 Ala(GCT)LCDR3 F YM113 270-304 280-282 Val(GTG)- pKC-dhfr-chKR127BS-V89A R YM114292-258 Ala(GCG) F YM115 274-307 283-285 Gln(CAA)-pKC-dhfr-chKR127BS-Q90A R YM116 294-259 Ala(GCA) F YM117 277-310 286-288Gly(GGT)- pKC-dhfr-chKR127BS-G91A R YM118 296-265 Ala(GCT) F YM119281-310 289-291 Thr(ACA)- pKC-dhfr-chKR127BS-T92A R YM120 302-266Ala(GCA) F YM121 282-313 292-294 His(CAT)- pKC-dhfr-chKR127BS-H93A RYM122 304-271 Ala(GCT) F YM111 286-314 295-297 Phe(TTT)-pKC-dhfr-chKR127BS-F94A R YM112 307-274 Ala(GCT) F YM123 286-317 298-300Pro(CCT)- pKC-dhfr-chKR127BS-P95A R YM124 308-278 Ala(GCT) F YM125292-319 301-303 Gln(CAG)- pKC-dhfr-chKR127BS-Q96A R YM126 311-279Ala(GCG) F YM127 294-320 304-306 Thr(ACG)- pKC-dhfr-chKR127BS-T97A RYM128 313-282 Ala(GCG)

TEST EXAMPLE 3 Measurement of Affinity to Antigen of Light Chain Mutant

COS7 cell was transfected with each of the light chain mutants preparedin Example 5 and the plasmid expressing chimeric heavychain(pcDdA-chKR127HC) to produce an antibody. The antibody obtained wasmeasured for its affinity to antigen in accordance with the methoddescribed in Test Example 1.

Table 6 shows the results obtained for the mutants and pdDA-chKR127HCcontaining wildtype chimeric KR127 heavy chain. TABLE 6 K_(D) CDR mutant(nM) L1 S26A  6.49 ± 0.244 Q27A 14.2 ± 2.29 S27aA 37.9 ± 6.66L27bA >10000 L27cA  36.8 ± 11.01 Y27dA 1032.7 ± 56.1  S27eA >10000N28A >10000 G29A 23.94 ± 2.62  K30A >10000 T31A 13.19 ± 1.98 Y32A >10000 N34A >10000 L2 L50A 159.4 ± 21.37 V51A 37.00 ± 10.33 S52A14.08 ± 0.509 K53A 7.928 ± 0.976 L54A 12.57 ± 2.453 D55A 225.2 ± 2.970S56A 12.95 ± 0.367 L3 V89A 121.2 ± 4.62  Q90A >10000 G91A >10000 T92A74.2 ± 2.90 H93A 54.5 ± 4.48 F94A >10000 P95A >10000 Q96A 293.6 ± 7.13 T97A 17.3 ± 2.56

As shown in Table 6, the affinities to antigen of the mutants obtainedby replacing the Leu27b, Tyr27d, Ser27e, Asn28, Lys30, Tyr32, and Asn34of LCDR1; Leu50 and Asp55 of LCDR2; and Val89, Gln90, Gly91, Thr92,His93, Phe94, Pro95, and Gln96 of LCDR3 with alanine, respectively, weremore than 3 times lower than that of the wild type. Therefore, theseresidues was determined as SDR.

EXAMPLE 6 Preparation of Humanized Heavy Chain by SDR-Grafting Method

A humanized heavy chain was prepared using DP7-JH4, a human heavy chainconstructed by combining human immunoglobulin germline VH gene segmentDP7 (Tomlinson et al., J. Mol. Biol., 227, 776-798, 1992) having anamino acid sequence similar to KR127 heavy chain variable regions andhuman immunoglobulin germline JH4 segment (Ravetch et al., Cell, 27,583-591 (1981)).

The Trp33 and Asn35 in HCDR1 of the KR127 were grafted into the DP7-JH4.The Met34 in HCDR1 of the KR127 is identical to that of DP7-JH4.Further, to inhibit lowering the affinity to antigen, Tyr32 in HCDR1 ofthe KR127 was replaced with alanine of HCDR1 of a human antibody (GenBank data base 75023 (SAWMN)).

The Arg50 and Tyr52 in HCDR2 of the KR127 were grafted onto the DP7-JH4.The Pro52a in HCDR2 of the KR127 is identical to that of DP7-JH4.

The Asp95, Tyr96, Arg97, Val98, and Arg102 of HCDR3 were grafted intoDP7-JH4.

Further, Ala71 and Lys73 of FR 3 (framwork region 3) in the heavy chainvariable region of KR127 antibody which affects the conformation of CDRloops were grafted thereto.

Then, PCR reaction was carried out using primers Ryu166 of SEQ ID NO: 23and Hur37 of SEQ ID NO: 24 according to the method described in Example3 to obtain a humanized heavy chain variable region gene, HuKR127VH-VII.Ryu 166: 5′-GGA TTT GTC TGC AGT CAT TGT GGC TCT GCC CTG GAA CTT-3′ Hur37: 5′-GAC AAA TCC ACG AGC ACA GTC TAC ATG-3′

The base sequence of the humanized heavy chain variable region gene wasdetermined by DNA sequence analysis (FIG. 2). Then, the gene was cleavedwith EcoRI and ApaI and inserted at the EcoRI/ApaI section of vectorpdDdA-chKR127HC to obtain pHuKR127HC.

A humanzied antibody was prepared by combining humanized heavy chainthus obtained and the humanized antibody HZKR127I light chain describedin Korean Patent No. 246128 and measured the affinity to antigen wasnumbered according to the method described in Test Example 2. Humanizedantibody HZKR127I was used as a control.

The affinity to antigen of the humanized antibody of about 1.5×10⁻¹⁰ Mwas about 50 times higher than that of HZKR127I, about 8.2×10⁻⁹ M.

EXAMPLE 7 Preparation of Humanized Light Chain by SDR-Grafting Method

A humanized light chain was prepared using DP7-JH4, a human light chianconstructed by combining human immunoglobulin germline VK gene segmentDPK12 (Cox et al., Eur. J immunol., 24, 827-836 (1994)) having an aminoacid sequence similar to KR127 light chain variable regions and humanimmunoglobulin germline JK4 segment (Hieter et al., J. Biol. Chem., 257,1516-1522 (1982)).

The Tyr27d, Asn28 and Asn34 in LCDR1 of KR127 were grafted into theDPK12-JK4. The amino acid residues at position 27b, 27e, 30 and 32 ofDP7 is identical to those of KR127 light chain.

The Leu50 and Asp55 in LCDR2 of KR127 were grafted into the DPK12-JK4gene.

The Val89, Gly91, Thr92, His93, Phe94, and Gln96 in LCDR3 of KR127 weregrafted into the DPK12-JK4. The residues at positions 90 and 95 of DP7is identical to those of KR127.

Further, Leu36 and Arg46 of FR 2 in the light chain variable region ofKR127 antibody (which acts on interaction with heavy chain or CDR) weregrafted thereto.

Then, PCR reaction was carried out using primers Ryu118 of SEQ ID NO: 25and Ryu119 of SEQ ID NO: 26 according to the method described in Example3 to prepare a humanized light chain variable region gene, HuKR127VH-IV.Ryu 118: 5′-CTG TGG AGG CTG GCC TGG CTT CTG TAA TAA CCA-3′ Ryu 119:5′-GGC CAG CCT CCA CAG CTC CTA ATC TAT CTG-3′

The base sequence of the humanized light chain variable region gene wasdetermined by DNA sequence analysis (see HZIV of FIG. 4). Then, the genewas cleaved with HindIII and BsiWI and inserted at the HindIII/BsiWIsection of vector pKC-dhfr-chKR127BS to obtain pHuKR127KC.

A humanized antibody was prepared by combining humanized light chainthus obtained and the humanized antibody HZKR127I heavy chain describedin Korean Patent No. 246128 and its affinity to antigen was measuredaccording to the method described in Test Example 2. Humanized antibodyHZKR127I was used as a control.

The affinities to antigen of the humanized antibody of about 8.4×10⁻⁹ Mwas similar to that of HZKR127I, about 8.2×10⁻⁹ M.

EXAMPLE 8 Preparation of Humanized Antibody and Measurement of theAffinity to Antigen

To prepare a plasmid containing humanized heavy chain plasmid pHuKR127HCand humanized light chain plasmid pHuKR127KC, the EcoRI/ApaI fragmentcontaining humanized heavy chain variable region gene of pHuKR127HC andthe HindIII/BsiWI fragment containing humanized light chain variableregion gene of pHuKR127KC were inserted at the EcoRI/ApaI andHindIII/BsiWI sections of vector pdCMV-dhfrC-HAV6 (KCTC 10028BP),respectively, to obtain plasmid pdCMV-dhfrC-HuKR127 (FIG. 6). E. coliDH5 α was transformed with the plasmid thus obtained and the transformedE. coli DH5α/pdCMC-dhfrC-HuKR127 was deposited on Mar. 13, 2002 with theKorean Collection for Type Cultures(KCTC)(Address: Korea ResearchInstitute of Bioscience and Biotechnology(KRIBB), #52, Oun-dong,Yusong-ku, Taejon, 305-333, Republic of Korea) under the accessionnumber, KCTC 10198BP, in accordance with the terms of Budapest Treaty onthe International Recognition of the Deposit of Microorganism for thePurpose of Patent Procedure.

To prepare cell line expressing the humanized antibody, dhfr-defectedCHO (chinese hamster ovary) cells were transformed with plasmidpdCMV-dhfrC-HuKR127 as follows:

CHO cells (ATCC CRL 9096) were seeded to DMEM/F12 media (GIBCO)containing 10% fetal bovine serum and subcultured in an incubator at 37°C. under an atmosphere of 5% CO₂. 5×10⁵ cells thus obtained were seededto the same media and incubated at 37° C. overnight, followed by washing3 times with OPTI-MEMI solution (GIBCO).

Meanwhile, 5 μg of the plasmid pdCMV-dhfrC-HuKR127 was diluted in 500 μlof OPTI-MEMI solution. 25 μl of Lipofectamine was diluted in 500 μl ofthe same solution. The resulting solutions were added to a 15 ml tube,mixed, and then, kept at room temperature for more than 15 minutes.Then, 2 ml of OPTI-MEM I was added to by DNA-Lipofectamine mixture andthe resulting solution was distributed evenly on the COS7 cells to bekept in a 5% CO₂ incubator at 37° C. for 6 hours. Added thereto was 3 mlof DMEM/F12 containing 20% fetal bovine serum and cultured for 48 hours.

Then, CHO cells were taken up with trypsin and cultured in a-MEMmedia(GIBCO) of 10% dialyzed fetal bovine serum containing G418 (GIBCOBRL, 550 mg/l) for 2 weeks. After confirming of antibody-producingability of the transformed clone, the clone was cultured in a-MEM mediaof 10% dialyzed fetal bovine serum containing 20 nM MTX to induceamplification of gene.

Cell line CHO/HuKR127 having the highest antibody-productivity wasselected from the clones and deposited on Mar. 13, 2002 with the KoreanCollection for Type Cultures(KCTC) under the accession number, KCTC10199BP, in accordance with the terms of Budapest Treaty on theInternational Recognition of the Deposit of Microorganism for thePurpose of Patent Procedure.

To measure the affinity to antigen of the humanized antibody HuKR127,CHO cell line thus obtained was mass cultured in a serum-absence media(CHO-SFMII, GIBCO) and subjected to protein G-shepharose 4B column(Pharmacia). Then, the antibody absorbed on the column was eluted with0.1 M glycine solution (pH 2.7) and neutralized with 1.0 M tris solution(pH 9.0), followed by dialyzing in PBS buffer (pH 7.0). Further, theaffinity to antigen of the purified antibody was determined by thecompetitive ELISA method described in Test Example 2 and compared withthat of a control, humanized HuKR127I. The result was shown in FIG. 7.

As shown in FIG. 7, the affinity to antigen of the humanized antibody ofthe present invention of 1.6×10⁻¹⁰ M was about 50 times higher than8.2×10⁻⁹ M3 of the control group.

EXAMPLE 9 Confirmation of Immune-Response Induction of HumanizedAntibody

To confirm whether the humanized antibody of the present invention(HuKR127) prevents HAMA response, an analysis was conducted according tothe TEPITOPE method (Sturniolo et al., Nature Biotechnology, 17,555-561, 1999) to examine whether a peptide sequences which can bind toMHC (major histocompatibility complex) class II exists in the heavy andlight chain variable regions of the humanized antibody.

Tables. 7 and 8 show the results of such analysis for MHC classII-binding peptide sequences in the heavy chain variable regions ofHuKR127 and the light chain variable regions of HuKR127, respectively.TABLE 7 HzKR127I HuKR127 antiboby peptide MHC class II peptide MHC classII MHC class II- LVQSGAEVV DRB1_0306 LVQSGAEVK 0 binding DRB1_0307DRB1_0308 DRB1_0311 DRB1_0421 DRB1_0701 DRB1_0703 VKPGASVKV DRB1_0102KKPGASVKV 0 FSSSWMNWV DRB1_0703 FTSAWMNWV 0 WIGRIYPGD DRB1_0801WMGRIYPSG 0 DRB1_0817 FQGKATLTA DRB1_0401 FQGRVTMTA DRB1_0305 DRB1_0402DRB1_0401 DRB1_0405 DRB1_0402 DRB1_0408 DRB1_0408 DRB1_0421 DRB1_0426DRB1_0426 DRB1_0801 DRB1_0801 DRB1_0802 DRB1_0802 DRB1_0804 DRB1_0804DRB1_0806 DRB1_0806 DRB1_0813 DRB1_0813 DRB1_0817 DRB1_0817 DRB1_1101DRB1_1101 DRB1_1114 DRB1_1102 DRB1_1120 DRB1_1104 DRB1_1128 DRB1_1106DRB1_1302 DRB1_1114 DRB1_1305 DRB1_1120 DRB1_1307 DRB1_1121 DRB1_1321DRB1_1128 DRB1_1323 DRB1_1302 DRB1_1502 DRB1_1305 DRB1_1307 DRB1_1311DRB1_1321 DRB1_1322 DRB1_1323 YWGQGTLVT DRB1_0401 RWGQGTLVT 0 DRB1_0405DRB1_0421 DRB1_0426 IGRIYPGDG DRB5_0101 MGRIYPSGG DRB1_0404 DRB5_0105DRB1_0405 DRB1_0410 DRB1_0423 YAQKFQGKA DRB1_0802 YAQKFQGRV 0 VYFCAREYDDRB1_1304 VYYCAREYR DRB1_0301 YWGQGTLVT DRB1_0401 RWGQGTLVT 0 DRB1_0405DRB1_0421 DRB1_0426 total 50 26 

TABLE 8 HzkR127I HuKR127 antiboby peptide MHC class II peptide MHC classII a MHC class II- ILMTQTPLS DRB1_0301 IVMTQTPLS 0 binding DRB1_0305DRB1_0306 DRB1_0307 DRB1_0308 DRB1_0309 DRB1_0311 DRB1_0401 DRB1_0402DRB1_0404 DRB1_0405 DRB1_0408 DRB1_0410 DRB1_0421 DRB1_0423 DRB1_0426DRB1_0804 DRB1_1101 DRB1_1102 DRB1_1104 DRB1_1106 DRB1_1107 DRB1_1114DRB1_1121 DRB1_1128 DRB1_1301 DRB1_1304 DRB1_1305 DRB1_1307 DRB1_1311DRB1_1321 DRB1_1322 DRB1_1323 DRB1_1327 DRB1_1328 LMTQTPLSL DRB1_0101VMTQTPLSL 0 DRB1_0102 DRB1_1304 WLLQKPGQS DRB1_0101 WLLQKPGQP 0DRB1_0305 DRB1_0309 DRB1_0401 DRB1_0408 DRB1_0421 DRB1_0426 DRB1_0802DRB1_1101 DRB1_1107 DRB1_1114 DRB1_1120 DRB1_1128 DRB1_1302 DRB1_1305DRB1_1307 DRB1_1321 DRB1_1323 DRB5_0101 DRB1_0105 YYCVQGTHF DRB1_0101YYCVQGTHF DRB1_0101 DRB1_0701 DRB1_0701 DRB1_0703 DRB1_0703 DRB5_0101DRB5_0101 DRB5_0105 DRB5_0105 YCVQGTHFP DRB1_0401 YCVQGTHFP DRB1_0401DRB1_0421 DRB1_0421 DRB1_0426 DRB1_0426 b VGVYYCVQG DRB1_0806 VGVYYCVQGDRB1_0806 IYLVSKLDS DRB1_0301 IYLVSNRDS DRB1_0402 DRB1_0305 DRB1_0404DRB1_0306 DRB1_0405 DRB1_0307 DRB1_0408 DRB1_0308 DRB1_0410 DRB1_0309DRB1_0423 DRB1_0311 DRB1_0804 DRB1_0405 DRB1_1102 DRB1_0410 DRB1_1104DRB1_0801 DRB1_1106 DRB1_0802 DRB1_1114 DRB1_0804 DRB1_1121 DRB1_0806DRB1_1301 DRB1_0813 DRB1_1307 DRB1_0817 DRB1_1311 DRB1_1101 DRB1_1322DRB1_1102 DRB1_1323 DRB1_1104 DRB1_1327 DRB1_1106 DRB1_1328 DRB1_1107DRB5_0101 DRB1_1114 DRB5_0105 DRB1_1120 DRB1_1121 DRB1_1128 DRB1_1301DRB1_1302 DRB1_1304 DRB1_1305 DRB1_1307 DRB1_1311 DRB1_1321 DRB1_1322DRB1_1323 DRB1_1327 DRB1_1328 DRB1_1501 DRB1_1506 LIYLVSKLD DRB1_0806LIYLVSNRD DRB1_0401 DRB1_1304 DRB1_0404 DRB1_1321 DRB1_0405 DRB1_0408DRB1_0410 DRB1_0421 DRB1_0423 DRB1_0426 DRB1_1304 YLVSKLDSG 0 YLVSNRDSGDRB1_0309 total 106  40 

As can be seen from FIGS. 7 and 8, the number of the peptide sequence inthe humanized antibody HuKR127 which binds to MHC class II was fewerthan of that the HzKR127I. These results suggest that humanized antibodyHuKR127 of the present invention is expected to reduce HAMA response toa greater extent than HzKR127I.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. A process for preparing a humanized antibody comprising the steps of:(a) selecting a specificity determining residue (SDR) of thecomplementarity determining region (CDR) of murine monoclonal antibodyheavy chain and light chain variable regions; and (b) grafting said SDRto at least one of the corresponding amino acid sequences into humanantibody variable regions.
 2. The process of claim 1, wherein step (a)is conducted by replacing each the amino acid residues of CDR withalanine to produce transformants, selecting a transformant that haslower affinity to the human antigen (K_(D)) than the original murineantibody and determining the replaced 15 amino acid residue of saidtransformant as an SDR.
 3. The process of claim 2, wherein the CDR isselected from the group consisting of HCDR1 (aa 31-35), HCDR2(aa 50-65)and HCDR3(aa 95-102) of the heavy chain (SEQ ID NO: 2); and LCDR1(aa24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain (SEQ IDNO: 4) of the murine monoclonal antibody variable regions of hepatitis Bvirus pre-S1 antigen, selecting a transformant that has an affinity toantigen which is more than 3 times lower than the original murineantibody when replaced with alanine, determining the replaced amino acidresidue of said transformant as an SDR, and grafting said SDR to thecorresponding amino acid sequence in human antibody heavy chain andlight chain.
 4. The process of claim 3, which is characterized in thatthe at least one of Trp33, Met34, and Asn35 of HCDRI; Arg5O, Tyr52, andPro52a of HCDR2; and G1u95, Tyr96, and G1u98 of HCDR3 of the murinemonoclonal antibody KR127 heavy chain, is grafted to the correspondingamino acid sequences in human antibody heavy chain.
 5. The process ofclaim 4, which is characterized in that the at least one of thefollowing grafting steps is carried out: (a) the amino acid residue atposition 32 in HCDR1 of human antibody with alanine; (b) the amino acidresidue at position 97 in HCDR3 of human antibody with arginine oralanine; (c) the amino acid residue at position 98 in HCDR3 of human 5antibody with valine; and (d) the amino acid residue at position 102 inHCDR3 of human antibody with arginine or alanine.
 6. The process ofclaim 5, which is characterized in that the at least one of Trp33 andAsn35 of HCDRI; Arg50 and Tyr52 of HCDR2; and Arg95 and Tyr96 of HCDR3of the murine monoclonal antibody KR127 heavy chain, is grafted into thehuman antibody heavy chain DP7-JH4.
 7. The process of claim 6, which ischaracterized in that the amino acid residues of the Ala71 and Lys73 inFramework region 3 of the murine monoclonal antibody KR127 heavy chainvariable region, of are further grafted into the human antibody heavychain DP7-JH4.
 8. The process of claim 3, which is characterized in thatthe at least one of the Leu27b, Tyr27d, Ser27e, Asn28, Lys30, Tyr32 andAsn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Val89, Gln9O, Gly9l,Thr92, His93, Phe94, Pro95, and G1n96 of LCDR3 of the murine monoclonalantibody KR127 light chain, is grafted into the human antibody lightchain.
 9. The process of claim 8, which is characterized in that theTyr27d, Asn28, Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Val89,Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 of the murinemonoclonal antibody KR127 light chain, is grafted into the humanantibody light chain DPH12-JK4.
 10. The process of claim 8, which ischaracterized in that the Leu36 and Arg46 in Framework region 2 of themurine monoclonal antibody KR127 light chain variable region, arefurther grafted into the human antibody light chain DPH12-JK4.
 11. Ahumanized antibody prepared by the process of any one of claims 1 to 10,which has an affinity to antigen of higher than 8.2×10⁹ M and suppressesHAMA (human anti-mouse antibody) response to a greater extent than anantibody prepared according to CDR-grafting method.
 12. The humanizedantibody of claim 11, which has the amino acid sequence of SEQ ID NO: 2for the heavy chain variable region of HBV pre-S1 antigen.
 13. Thehumanized antibody of claim 11, which has the amino acid sequence of SEQID NO: 4 for the light chain variable region of HBV pre-S1 antigen. 14.A humanized antibody which is produced by CHO/HuKR127 (Accession No.:KCTC 10199BP).
 15. A DNA encoding the humanized antibody heavy chaincontaining the amino acid sequence of SEQ ID NO: 2 for the heavy chainvariable region of HBV pre-S1 antigen.
 16. The DNA of claim 15, whereinthe variable region has the nucleotide sequence of SEQ ID NO:
 1. 17. ADNA encoding the humanized antibody light chain containing the aminoacid sequence of SEQ ID NO: 4 for the light chain variable region of HBVpre-S1 antigen.
 18. The DNA of claim 17, wherein the variable region hasthe nucleotide sequence of SEQ ID NO:
 3. 19. An expression vectorpHuKR127HC comprising the DNA of claim 16 for expressing the humanizedantibody heavy chain for HBV pre-S1 antigen.
 20. An expression vectorpHuKR127KC comprising the DNA of claim 18 for expressing the humanizedantibody light chain for HBV pre-S1 antigen.
 21. An expression vectorpdCMV-dhfrC-HuKR127 comprising the nucleotide sequences of SEQ ID NO:1and SEQ ID NO:3 for expressing the humanized antibody light and heavychains for HBV pre-S1 antigen.
 22. An E. coli DH5α/pdCMV-dhfrC-HuKR127(Accession No.: KCTC 10198BP) transformed with the expression vector ofclaim
 21. 23. CHO cell line CHOIHuKR127 (Accession No.: KCTC 1010199BP)producing the humanized antibody of claim
 11. 24. A composition forpreventing or treating HBV infection comprising the humanized antibodyof claim 11.